Cholestanol derivative for combined use

ABSTRACT

The invention provides a cancer chemotherapeutic agent which has fewer side effects and excellent efficacy. The cancer chemotherapeutic agent of the invention includes a cholestanol derivative represented by formula (1): 
     
       
         
         
             
             
         
       
     
     (wherein G represents GlcNAc-Gal-, GlcNAc-Gal-Glc-, Fuc-Gal-, Gal-Glc-, Gal-, or GlcNAc-) or a cyclodextrin inclusion compound thereof, and an anti-cancer agent.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a chemotherapeutic agent for cancer(hereinafter referred to as a “cancer chemotherapeutic agent”) and, moreparticularly, to a cancer chemotherapeutic agent employing a cholestanolderivative and an anti-cancer agent in combination.

2. Background Art

A variety of anti-cancer agents used in chemotherapy for cancer, whichis one mode of cancer therapy, have hitherto been developed andclassified based on structure, action mechanism, etc. However, theefficacy of such an anti-cancer agent employed as a single agent isunsatisfactory. Instead, multi-drug therapy employing a plurality ofanti-cancer agents has been predominantly carried out in recent yearsfrom the viewpoint of suppressing adverse side effects, and the effecacyof multi-drug therapy has been recognized.

Under such circumstances, both of the development of new anti-cancercombination chemotherapy, which has fewer adverse side effect and higherefficacy than conventional chemotherapies, and the development of newchemotherapeutic agents for use in the chemotherapy are desired.

Meanwhile, a cholestanol derivative, in which a sugar chain such asGlcNAc-Gal-, GlcNAc-Gal-Glc-, Fuc-Gal-, Gal-Glc-, Gal-, or GlcNAc- isbonding to cholestanol (the compound that the double bond in the B ringof the cholesterol is saturated), were previously found to haveexcellent anti-tumor activity. JP-A-2000-191685, JP-A-1999-60592, WO2005/007172 (pamphlet), and WO 2007/026869 (pamphlet) disclose theeffects of such cholestanol derivatives.

However, no cases have been reported in which the aforementionedcholestanol derivatives and another anti-cancer agent are employed incombination.

SUMMARY OF THE INVENTION

Thus, the present invention is directed to provision of a cancerchemotherapeutic agent which has fewer side effects and excellentefficacy.

In view of the foregoing, the present inventors have carried outextensive studies, and have found that a remarkably potentiatedanti-cancer effect can be attained through employment, in combination,of a cholestanol derivative represented by formula (1) or a cyclodextrininclusion compound thereof and a known chemotherapeutic agent(anti-cancer agent), and thus the combined use of these pharmaceuticalagents in cancer chemotherapy is very useful.

Accordingly, the present invention is directed to the following (1) to(10).

(1) A cancer chemotherapeutic agent comprising, in combination, acholestanol derivative represented by formula (1):

(wherein G represents GlcNAc-Gal-, GlcNAc-Gal-Glc-, Fuc-Gal-, Gal-Glc-,Gal-, or GlcNAc-) or a cyclodextrin inclusion compound thereof, and ananti-cancer agent.(2) A cancer chemotherapeutic agent according to (1) above, wherein, informula (1), G is GlcNAc-Gal- or GlcNAc-.(3) A cancer chemotherapeutic agent according to (1) or (2) above,wherein the anti-cancer agent is one or more species selected from thegroup consisting of a taxane anti-cancer agent, a platinum complexanti-cancer agent, a pemetrexed compound, and fluorouracil.(4) A cancer chemotherapeutic agent according to (3) above, wherein theanti-cancer agent is one or more species selected from the groupconsisting of Paclitaxel, Docetaxcel, Pemetrexed, 5-FU, Cisplatin,Oxaliplatin, Cyclophosphamide, and Irinotecan.(5) A cancer chemotherapeutic agent according to any of (1) to (4)above, which is a compounding agent.(6) A cancer chemotherapeutic agent according to any of (1) to (4)above, which is in the form of a kit including a drug containing acholestanol derivative and a drug containing an anti-cancer agent.(7) A cancer chemotherapeutic agent according to (6) above, wherein thedrug containing a cholestanol derivative is a liposomal formulation.(8) Use, in combination, of a cholestanol derivative represented byformula (1):

(wherein G represents GlcNAc-Gal-, GlcNAc-Gal-Glc-, Fuc-Gal-, Gal-Glc-,Gal-, or GlcNAc-) or a cyclodextrin inclusion compound thereof and ananti-cancer agent, for producing a cancer chemotherapeutic agent.(9) A cancer chemotherapy, characterized by comprising administering, incombination, a cholestanol derivative represented by formula (1):

(wherein G represents GlcNAc-Gal-, GlcNAc-Gal-Glc-, Fuc-Gal-, Gal-Glc-,Gal-, or GlcNAc-) or a cyclodextrin inclusion compound thereof and ananti-cancer agent, to a patient in need thereof.(10) A cancer chemotherapy according to (9) above, wherein thecholestanol derivative or a cyclodextrin inclusion compound thereof andthe anti-cancer agent are administered to a patient in need thereofsimultaneously, or separately at intervals.

Through employment of the cancer chemotherapeutic agent and the cancerchemotherapy according to the present invention, prevention andtreatment of cancer can be realized with safety and higher efficacy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the cell proliferation inhibitory effects ofCDDP, GC-CD, and CDDP+GC-CD on colon 26 cells;

FIG. 2 is a graph showing the cell proliferation inhibitory effects ofCDDP, GC-CD, and CDDP+GC-CD on MKN45 cells, NCIH226 cells, or colo201cells;

FIG. 3 is a graph showing the cell proliferation inhibitory effects ofCDDP, GGC-CD, and CDDP+GGC-CD on colon26 cells, MKN45 cells, NCIH226cells, or colo201 cells;

FIG. 4 is a graph showing the anti-tumor effect of single administrationof CDDP, GC-CD, and CDDP+GC-CD against peritoneal dissemination causedby colon26 cells intraperitoneally inoculated in mice;

FIG. 5 is a graph showing the anti-tumor effect of multipleadministration of CDDP, GC-CD, and CDDP+GC-CD against peritonealdissemination caused by colon26 cells intraperitoneally inoculated inmice;

FIG. 6 is a graph showing the anti-tumor effect of single, delayedadministration of CDDP, GC-CD, and CDDP+GC-CD against peritonealdissemination caused by colon26 cells intraperitoneally inoculated inmice, after confirmation of peritoneal dissemination on the mesotheliumof mice;

FIG. 7 is a graph showing the survival rate of mice to which colon26cells were intraperitoneally inoculated, upon single administration ofCDDP, GC-CD, or CDDP+GC-CD (single administration of CDDP and doubleadministration of GC-CD, respectively);

FIG. 8 is a graph showing the effect of suppressing or reducing thetumor growth to which colon26 cells were subcutaneously inoculated inmice, upon single administration of CDDP, GGC-CD, or CDDP+GGC-CD;

FIG. 9 is a graph showing the effect of inhibiting metastatis of colon26cells to the lung, upon single administration of CDDP, GC-CD, GGC-CD,CDDP+GC-CD, and CDDP+GGC-CD;

FIG. 10-A is a graph showing the cell proliferation inhibitory effectsof an anti-cancer agent (5-FU, PTX, DTX, or CPT), GC-CD, and theanti-cancer agent+GC-CD on colon 26 cells; and

FIG. 10-B is a graph showing the cell proliferation inhibitory effectsof an anti-cancer agent (L-OHP or CPA), GC-CD, and the anti-canceragent+GC-CD on colon 26 cells.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The specific cholestanol derivatives represented by formula (1) andemployed in the present invention are all known compounds.

Among the cholestanol derivatives which are represented by formula (1)and in which G is GlcNAc-Gal-, G is preferably GlcNAcβ1,3-Galβ- orGlcNAcβ1,4-Galβ-. Among the cholestanol derivatives (1) in which G isGlcNAc-Gal-Glc-, G is preferably GlcNAcβ1,3-Galβ1,4-Glc-. Among thecholestanol derivatives (1) in which G is Fuc-Gal-, G is preferablyFucα1,3Gal-. Among the cholestanol derivatives (1) in which G isGal-Glc-, G is preferably Galβ1,4Glcβ-. Among the cholestanolderivatives (1) in which G is Gal-, G is preferably Galβ-. Among thecholestanol derivatives (1) in which G is GlcNAc-, G is preferablyGlcNAcβ—.

Of these, species in which G is GlcNAc-Gal- and GlcNAc- are morepreferred, with those in which G is GlcNAcβ1,4-Galβ- and GlcNAcβ- beingstill more preferred.

The aforementioned cholestanol derivatives may be produced through amethod, for example, disclosed in the aforementioned Patent Documents ora similar method.

The cholestanol derivative represented by (1) readily forms an inclusioncomplex with a cyclodextrin or a derivative thereof. Thus, thecholestanol derivative employed in the present invention may be acyclodextrin inclusion compound thereof. In formation of such inclusioncompounds, the size of the guest molecule to be included, Van der Waalsinteraction between the guest molecule and cyclodextrin, and hydrogenbond between the hydroxyl groups of cyclodextrin and the guest moleculemust be taken into consideration. Therefore, insoluble guest compoundsdo not always form the corresponding inclusion compounds. However, thecholestanol derivative of the present invention can form good inclusioncomplexes with cyclodextrin.

Examples of the cyclodextrin forming the cyclodextrin inclusion compoundof the present invention include cyclodextrins such as α-cyclodextrin,β-cyclodextrin, and γ-cyclodextrin; and cyclodextrion derivatives suchas methyl-β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin,monoacetyl-β-cyclodextrin, and 2-hydroxypropyl-γ-cyclodextrin. Of these,2-hydroxypropyl-β-cyclodextrin is preferred for obtaining improvedsolubility.

The cyclodextrin inclusion compound may be prepared through, forexample, the following procedure: an aqueous solution of a cyclodextrinor a derivative thereof having an appropriate concentration (e.g., 20 to40%) is prepared, and the cholestanol derivative of the presentinvention is added to the aqueous solution, followed by stirring of theresultant mixture.

No particular limitation is imposed on the concentration of the solutionof the cholestanol derivative (1), so long as the cholestanol derivativecan form an inclusion compound with cyclodextrin. Generally, theconcentration is about 1 to about 50 mass %, preferably about 10 toabout 30 mass %.

The thus-produced cyclodextrin inclusion compound is highlywater-soluble and, therefore, effectively exhibits the effect of theguest in vivo. Another advantage of the cyclodextrin inclusion compoundis to ensure consistent in vitro test results.

Alternatively, the cholestanol derivative (1) may be prepared into aliposomal formulation, whereby the cholestanol derivative can be moreeffectively delivered to the action expression site. Another advantageof the cyclodextrin inclusion compound is to ensure consistent in vitrotest results.

Preferably, the liposomal formulation includes the cholestanolderivative of the present invention, a membrane component, and analiphatic or aromatic amine.

The cholestanol derivative content in the liposomal formulation ispreferably 0.3 to 2.0 mol, more preferably 0.8 to 1.5 mol, with respectto 1 mol of the membrane component.

The membrane component may be a phospholipid. Specific examples ofpreferably employed phospholipids include natural and syntheticphospholipids such as phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, and phosphatidic acid;mixtures thereof; and modified natural phospholipids such as aqueouslecithin. Examples of more preferred species include phosphatidylcholine(1α-dipalmitoylphosphatidylcholine (DPPC)).

The aliphatic or aromatic amine is employed mainly for positivelycharging the surface of lipid membrane. Examples of such amines includealiphatic amines such as stearylamine and oleylamine; and aromaticamines such as fluorenethylamine. Among them, stearlylamine isparticularly preferably employed.

Preferably, the amine is contained in an amount of 0.04 to 0.15 mol,more preferably 0.1 to 0.15 mol, with respect to 1 mol of membranecomponent (phospholipid).

In addition to the aforementioned components, if required, the liposomemay further contain a membrane structure stabilizer such as cholesterol,fatty acid, diacetyl phosphate, etc.

The aqueous solution employing for dispersing the membrane component ispreferably water, physiological saline, buffer, aqueous sugar solution,or a mixture thereof. Either an organic or an inorganic buffer may beused, so long as the buffer has a buffering action in the vicinity ofthe hydrogen ion concentration of body fluid. Examples of such buffersinclude a phosphate buffer.

No particular limitation is imposed on the method of preparing theliposomal formulation, and generally employed methods may be selected.Examples of the employable method include methods disclosed inJP-A-1982-82310, JP-A-1985-12127, JP-A-1985-58915, JP-A-1989-117824,JP-A-1989-167218, JP-A-1992-29925, and JP-A-1997-87168; a methoddisclosed in Methods of Biochemical Analysis (1988) 33, p. 337; or amethod disclosed in “Liposome” (published by Nankodo).

No particular limitation is imposed on the anti-cancer agent which isused in combination with the cholestanol derivative represented byformula (1) or a cyclodextrin inclusion compound thereof, and knowncancer chemotherapeutic agents may be used. Standard therapeutic agentswhich have been established with respect to the cancer of therapy targetare preferably employed.

Specific examples include alkylating agents such as Cyclophosphamide,Ifosfamide, Melphalan (L-PAM), Busulfan, and Carboquione; metabolismantagonists such as 6-Mercaptopurine (6-MP), Methotrexate (MTX),5-Fluorouracil (5-FIT), Tegafur, Enocitabine (BHAC), and pemetrexedcompounds (Pemetrexed, Alimta, MTA), etc.; carcinostatic antibioticssuch as Actinomycin D, Daunorubicin, Bleomycin, Peplomycin, Mitomycin C,Aclarubicin, and Neocarzinostatin (NCS); plant alkaloids such asVincristine, Vindesine, Vinblastine, taxane anti-cancer agents (Taxotere(Docetaxel) and Taxol (Paclitaxel, TXL), etc.), and Irinotecan (CPT-11);and platinum compounds such as Cisplatin (CDDP), and Carboplatin,Oxaliplatin (L-OHP). These anti-cancer agents may be used singly or incombination of two or more species.

As shown in the Examples described hereinbelow, when the cholestanolderivative represented by formula (1) or a cyclodextrin inclusioncompound thereof is used in combination with an anti-cancer agent,proliferation of cancer cells of various types are strongly suppressed,as compared with the case of administration of only each agent.Therefore, this combined chemotherapy can drastically enhancetherapeutic efficacy and mitigation of adverse side effects, and apharmaceutical product containing these ingredients is a useful cancerchemotherapeutic agent.

No particular limitation is imposed on the cancer which can beeffectively treated by administering the cancer chemotherapeutic agentof the present invention. Examples of the target cancer includemalignant tumors such as gastric cancer, large bowel cancer, pancreaticcancer, uterus cancer, ovaria cancer, lung cancer, gallbladder cancer,esophageal cancer, liver cancer, breast cancer, mesothelioma, andprostatic cancer.

The form of the cancer chemotherapeutic agent of the present inventionmay be a compounding agent in which the aforementioned ingredients aremixed at an appropriate ratio, each at an effective amount, to form asingle dosage form (single-formulation type), or may be a kit thatconsists of the respective dosage form of the aforementionedingredients, each of which is formed independently including eacheffective amount, and that enables the dosage forms to be administeredsimultaneously or separately at intervals (double-formulation type).

Similar to general pharmaceutical formulations, no particular limitationis imposed on the dosage form of the above-described formulation, andthe form may be any of the solid form such as tablet, liquid form suchas injection, dry powder dissolving before use, etc.

No particular limitation is imposed on the administration route of theformulation, and an appropriate route may be determined depending on thedosage form of the agents. For example, an injection solution may beadministered intravenously, intramuscularly, subcutaneously,intradermally, or interperitoneally, and a solid form may beadministered orally or enterally.

The formulation may be prepared through a known method in the art. Allpharmaceutically acceptable carriers (excipients or diluents such as afiller, a bulking agent, and a binder) generally employed in the art mayalso be employed.

For example, a peroral solid form may be prepared through mixing thedrug ingredients of the present invention with a excipient, and with anoptional binder, disintegrant, lubricant, colorant, flavoring agent,deodorant, etc., and forming the mixture into tablets, coated-tablets,granules, powder, capsules, etc. through a method known in the art.These additive may be those generally employed in the art. Examples ofthe excipient include lactose, sucrose, sodium chloride, glucose,starch, calcium carbonate, kaolin, microcrystalline cellulose, andsilicic acid. Examples of the binder include water, ethanol, propanol,simple syrup, liquid glucose, liquid starch, liquid gelatin,carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch,methyl cellulose, ethyl cellulose, shellac, calcium phosphate, andpolyvinylpyrrolidone. Examples of the disintegrant include dry starch,sodium alginate, agar powder, sodium hydrogencarbonate, calciumcarbonate, sodium lauryl sulfate, monoglyceryl stearate, and lactose.Examples of the lubricant include purified talc, stearate salts, borax,and polyethylene glycol. Examples of the flavoring agent includesucrose, orange peel, citric acid, and tartaric acid.

An oral liquid formulation may be prepared by mixing the drugingredients of the present invention with a flavoring agent, buffer,stabilizer, deodorant, etc., and forming the mixture into internalliquid agent, syrup, elixir, etc. through a method known in the art. Theflavoring agent employed in the preparation may be any of theaforementioned members. Examples of the buffer include sodium citrate.Examples of the stabilizer include traganth, gum arabic, and gelatin.

Injection solutions may be prepared by mixing the drug ingredients ofthe present invention with additives such as a pH-regulator, buffer,stabilizer, tonicity agent, and local anesthetic agent, etc., andforming the mixture through a method known in the art, to therebyprovide subcutaneous, intramuscular, and intravenous injection liquids.Examples of the pH-regulator and buffer include sodium citrate, EDTA,thioglycolic acid, and thiolactic acid. Examples of the local anestheticinclude procaine hydrochloride and lidocaine hydrochloride. Examples ofthe tonicity agent include sodium chloride and glucose.

Suppositories may be prepared by mixing the drug ingredients of thepresent invention with a carrier for formulation known in the art suchas polyethylene glycol, lanolin, cacao butter, and fatty acidtriglyceride, and with an optional surfactant such as Tween (registeredtrademark), and forming the mixture into suppositories through a methodknown in the art.

Ointments may be prepared by mixing the drug ingredients of the presentinvention with optional additives generally employed in the art such asa base, stabilizer, moisturizer, and preservatives, and forming themixture into ointments through a method known in the art. Examples ofthe base include liquid paraffin, white petrolatum, white beeswax,octyldodecyl alcohol, and paraffin. Examples of the preservative includemethyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, and propylp-hydroxybenzoate.

Cataplasms may be prepared by applying the aforementioned ointment, gel,cream, paste, etc., to a generally employed support through a routinemethod. Examples of appropriate supports include woven and nonwovenfabric made of cotton, staple fiber, or chemical fiber, and film andfoamed sheet made of soft vinyl chloride, polyethylene, polyurethane,etc.

Generally, the formulation is preferably prepared so as to have acholestanol derivative content and an anti-cancer agent content of0.0001 to 80 wt. % (as effective ingredient).

When the cancer chemotherapeutic agent of the present invention isprovided as a kit, the kit may be designed to pack independently therespective dosage form including separately the cholestanol derivativerepresented by formula (1) or a cyclodextrin inclusion compound thereofand an anti-cancer agent, each of which have been prepared in the abovemanner, and to be used each pharmaceutical formulation taken separatelyfrom the corresponding respective package before use. Alternatively,each pharmaceutical formulation may be held in a package suitable foreach time of combined administration.

The dose of the cancer chemotherapeutic agent of the present inventionvaries depending on the body weight, age, sex, symptoms of a patient inneed thereof, route and frequency of administration to a patient in needthereof, etc. Generally, for example, the daily dose for an adult isabout 0.1 to 30 mg/kg as the cholestanol derivative (1), preferably 3 to10 mg/kg. The dose of the anti-cancer agent may fall within a rangeestablished with respect to the agent, or may be lower than that range.

No particular limitation is imposed on the frequency of administration,and the agent may be administered once or several times a day. Singleadministration a day is preferred. When the kit is used, each of theformulation including separated drug ingredients may be administeredsimultaneously or intermittently.

EXAMPLES

The present invention will next be described in more detail by way ofexamples, which should not be construed as limiting the inventionthereto.

Example 1 Effect of Drug Addition on Inhibition of Cancer CellProliferation

Colon26 cells (derived from mouse colon cancer) were inoculated to a96-well plate (1×10⁴ cells/50 μL, 10% FCS-RPMI medium/well), andincubated at 37° C. for 16 hours. To each well, cisplatin (abbreviatedas “CDDP”) and/or a cyclodextrin inclusion compound (abbreviated as“GC-CD”) of a cholestanol derivative in which G in formula (1) isGlcNAcβ-(abbreviated as “GC”) was added (multi-fold dilution byFCS(−)-medium: final concentration: ≦0.500 μM, 50 μL), followed byincubation at 37° C. for two days. GC-CD was prepared in accordance witha method disclosed in Example 1(2) in WO 2007/026869. Specifically, a40% aqueous solution of hydroxypropyl-β-cyclodextrin was prepared, andGC was added to the solution, followed by mixing with stirring (80° C.for 30 minutes), to thereby prepare GC-CD.

As a control, wells to which only FCS(−)-medium had been added wereemployed. Viable count was performed by means of a cell counting kit(product of Dojin).

Cell proliferation inhibition (CPI) rate (%) was calculated by thefollowing equation. FIG. 1 shows the results.

${{CPI}\mspace{14mu} {rate}\mspace{14mu} (\%)} = {\left( {1 - \frac{{treated}\mspace{14mu} {cells}\mspace{14mu} {OD}_{450 - 650}}{{untreated}\mspace{14mu} {cells}\mspace{14mu} {OD}_{450 - 650}}} \right) \times 100}$

Example 2 Effect of Inhibition of Proliferation of Various Cancer Cells

The procedure of Example 1 was repeated, except that colon26 cells werechanged to MKN45 (derived from human gastric cancer), NCIH226 (derivedfrom human lung cancer), and Colo201 (derived from human colon cancer).CPI rate (%) was determined in a similar manner. FIG. 2 shows theresults.

In Example 2, a cyclodextrin inclusion compound (abbreviated as“GGC-CD”) of a cholestanol derivative in which G in formula (1) isGlcNAcβ1,4-Galβ- (abbreviated as “GGC”) was also used. GGC-CD wasproduced in a manner similar to the method as the aforementioned GC-CDproduction method, except that the cholestanol compound was changed toGGC. CPI rate with respect to the cancer cells was determined. FIG. 3shows the results.

Example 3 Effect of Drug Addition on Inhibition of Cancer CellProliferation In Vivo

In the following Examples, Balb/c mice (6-weeks old, female) wereemployed as test animals.

(1) Colon26 cells (1×10⁴ cells/mouse) were intraperitoneally inoculatedto the mice (day 0). On the following day after inoculation (day 1),CDDP and/or GC-CD was adjusted with physiological saline (Otsuka normalsaline) to a concentration of interest, and CDDP, GC-CD, or CDDP+GC-CD(500 μL) was intraperitoneally administered to the mice, followed bybreeding. On day 19, mice were dissected, and the weight of themesentery and the greater omentum was measured. To the control group,only physiological saline (500 μL) was administered (n=10; 10mice/group).

FIG. 4 shows the results.

(2) Colon26 cells (1×10⁴ cells/mouse) were intraperitoneally inoculatedto the mice (day 0). On day 1, day 2, day 3, day 6, day 7, and day 8,CDDP and/or GC-CD was adjusted with physiological saline (Otsuka normalsaline) to a concentration of interest, and CDDP, GC-CD, or CDDP+GC-CD(500 μL) was intraperitoneally administered to the mice, followed bybreeding. On day 21, mice were dissected, and the weight of themesentery and the greater omentum was measured. To the control group,only physiological saline (500 μL) was administered (n=10; 10mice/group).

FIG. 5 shows the results.

(3) Colon26 cells (1×10⁴ cells/mouse) were intraperitoneally inoculatedto the mice (day 0). On day 7, CDDP and/or GC-CD was adjusted withphysiological saline (Otsuka normal saline) to a concentration ofinterest, and CDDP, GC-CD, or CDDP+GC-CD (500 μL) was intraperitoneallyadministered to the mice, followed by breeding. On day 18, mice weredissected, and the weight of the mesentery and the greater omentum wasmeasured. To the control group, only physiological saline (500 μL) wasadministered (n=10; 10 mice/group).

FIG. 6 shows the results.

Example 4 Anti-Tumor Effect by Drug Addition

Balb/c mice (6 weeks old, female) were employed as test animals. Colon26cells (1×10⁴ cells/mouse) were intraperitoneally inoculated to the mice(day 0). On day 2 and/or day 3, CDDP and/or GC-CD was adjusted withphysiological saline (Otsuka normal saline) to a concentration ofinterest, and CDDP (once, on day 2), GC-CD (twice, on day 2 and 3), orCDDP (once, on day 2)+GC-CD (twice, day 2 and 3) (500 μL) wasintraperitoneally administered to the mice, followed by breeding. Thesurvival duration (days) was counted to day 43. To the control group,only physiological saline (500 μL) was administered (n=10; 10mice/group).

FIG. 7 shows the results.

Example 5 Anti-Tumor Effect by Drug Addition

Balb/c mice (6 weeks old, female) were employed as test animals. Colon26cells (5×10⁴ cells/mouse) were subcutaneously inoculated to the mice(day 0). After confirmation that the tumor size reached about 4 mm (day7 to 10 after inoculation), CDDP and/or GGC-CD was adjusted withphysiological saline (Otsuka normal saline) to a concentration ofinterest, and CDDP, GGC-CD, or CDDP+GGC-CD (200 μL) was administered tothe mice through the tail vein, followed by breeding. Time-dependentchange in tumor size was monitored to day 21, and the correspondingtumor volume was determined. To the control group, only physiologicalsaline (200 μL) was administered (n=7; 7 mice/group).

FIG. 8 shows the results.

Example 6 Cancer Metastasis Inhibitory Effect by Drug Addition

Balb/c mice (6 weeks old, female) were employed as test animals. Colon26cells (5×10⁴ cells/mouse) were intraperitoneally inoculated to the mice(day 0). Immediately after inoculation, CDDP and/or GC-CD or GGC-CD wasadjusted with physiological saline (Otsuka normal saline) to aconcentration of interest, and CDDP, GC-CD (or GGC-CD), or CDDP+GC-CD(or GGC-CD) (200 μL) was administered to the mice through the caudalvein, followed by breeding. On day 14, mice were dissected, and thetumor nodes in the lungs were counted. To the control group, nosubstance was administered (n=10; 10 mice/group).

FIG. 9 shows the results.

Example 7 Effect of Drug Addition on Inhibition of Cancer CellProliferation

Colon26 cells (derived from mouse colon cancer) were inoculated to a96-well plate (1×10⁴ cells/50 μL, 10% FCS-RPMI medium/well), andincubated at 37° C. for 16 hours. To each well, well-known anti-cancersagent (Oxaliplatin (abbreviated as “L-OHP”), Fluorouracil (5-FU),Paclitaxel (TXL; abbreviated as “PTX”), Docetaxel (TXT; abbreviated as“DTX”), Irinotecan (CPT-11; abbreviated as “CPT”), or Cyclophosphamide(abbreviated as “CPA”) and/or a cyclodextrin inclusion compound(abbreviated as “GC-CD”) of a cholestanol derivative in which G informula (1) is GlcNAcβ- (abbreviated as “GC”) was added (multi-folddilution by FCS(−)-medium: final concentration: ≦500 μM, 50 μL),followed by incubation at 37° C. for two days. GC-CD was prepared inaccordance with a method disclosed in Example 1(2) in WO 2007/026869.Specifically, a 40% aqueous solution of hydroxypropyl-β-cyclodextrin wasprepared, and GC was added to the solution, followed by mixing withstirring (80° C. for 30 minutes), to thereby prepare GC-CD.

As a control, wells to which only FCS(−)-medium had been added wereemployed. Viable count was performed by means of a cell counting kit(product of Dojin).

Cell proliferation inhibition (CPI) rate (%) was calculated by thefollowing equation. FIG. 10 (FIGS. 10-A and 10-B) shows the results.

${{CPI}\mspace{14mu} {rate}\mspace{14mu} (\%)} = {\left( {1 - \frac{{treated}\mspace{14mu} {cells}\mspace{14mu} {OD}_{450 - 650}}{{untreated}\mspace{14mu} {cells}\mspace{14mu} {OD}_{450 - 650}}} \right) \times 100}$

As described hereinabove, through employment, in combination, of thecholestanol derivative of the present invention or a cyclodextrininclusion compound thereof and an anti-cancer agent, proliferation ofvarious cancer cells is strongly inhibited, and synergistic effectand/or effect of potentiating anti-tumor action of a known anti-canceragent can be obtained.

1. A cancer chemotherapeutic agent comprising, in combination, acholestanol derivative represented by formula (1):

wherein G represents GlcNAc-Gal-, GlcNAc-Gal-Glc-, Fuc-Gal-, Gal-Glc-,or Gal- or a cyclodextrin inclusion compound thereof, and an anti-canceragent.
 2. The cancer chemotherapeutic agent according to claim 1,wherein, in formula (1), G is GlcNAc-Gal-.
 3. The cancerchemotherapeutic agent according to claim 1, wherein the anti-canceragent is one or more species selected from the group consisting of ataxane anti-cancer agent, a platinum complex anti-cancer agent, apemetrexed compound, and fluorouracil.
 4. The cancer chemotherapeuticagent according to claim 3, wherein the anti-cancer agent is one or morespecies selected from the group consisting of Paclitaxel, Docetaxcel,Pemetrexed, 5-FU, Cisplatin, Oxaliplatin, Cyclophosphamide, andIrinotecan.
 5. The cancer chemotherapeutic agent according to claim 1,which is a compounding agent.
 6. The cancer chemotherapeutic agentaccording to claim 1, which is in the form of a kit including a drugcontaining a cholestanol derivative and a drug containing an anti-canceragent.
 7. The cancer chemotherapeutic agent according to claim 6,wherein the drug containing a cholestanol derivative is a liposomalformulation.
 8. A method of producing a chemotherapeutic agent for thetreatment of cancer, by combining a cholestanol derivative representedby formula (1):

wherein G represents GlcNAc-Gal-, GlcNAc-Gal-Glc-, Fuc-Gal-, Gal-Glc-,or Gal- or a cyclodextrin inclusion compound thereof and an anti-canceragent.
 9. A method for cancer chemotherapy, comprising administering, incombination, a cholestanol derivative represented by formula (1):

wherein G represents GlcNAc-Gal-, GlcNAc-Gal-Glc-, Fuc-Gal-, Gal-Glc-,or Gal- or a cyclodextrin inclusion compound thereof and an anti-canceragent, to a patient in need thereof.
 10. The method for cancerchemotherapy according to claim 9, wherein the cholestanol derivative ora cyclodextrin inclusion compound thereof and the anti-cancer agent areadministered to a patient in need thereof simultaneously, or separatelyand intermittently.
 11. The method for cancer chemotherapy according toclaim 9, wherein, in formula (1), G is GlcNAc-Gal-.
 12. The method forcancer chemotherapy according to claim 9, wherein the anti-cancer agentis one or more species selected from the group consisting of a taxaneanti-cancer agent, a platinum complex anti-cancer agent, a pemetrexedcompound, and fluorouracil.
 13. The method for cancer chemotherapyaccording to claim 12, wherein the anti-cancer agent is one or morespecies selected from the group consisting of Paclitaxel, Docetaxcel,Pemetrexed, 5-FU, Cisplatin, Oxaliplatin, Cyclophosphamide, andIrinotecan.
 14. The method for cancer chemotherapy according to claim 9,wherein the drug containing a cholestanol derivative is a liposomalformulation.
 15. The method for cancer chemotherapy according to claim9, wherein the anticancer agent is Cisplatin.
 16. The cancerchemotherapeutic agent according to claim 1, wherein the anticanceragent is Cisplatin.